Research in epigenetics has led to the discovery of large multi-protein complexes that catalyze histone modifications and their removal, affecting nucleosomal remodeling that either promotes or interferes with transcriptional access to genes. One of these, the repressive CoREST complex, includes the enzymes LSD1 histone demethylase, HDAC1 (histone deacetylase 1) and the scaffolding protein CoREST1. The CoREST complex has not yet been well-characterized biochemically in its intact form. The goal of this project is to provide a deeper biochemical understanding of the CoREST complex. Through kinetic analysis of the CoREST complex with post-translationally modified peptide substrates, we expect to reveal cross-talk among histone modifications and the regulatory effects of protein-protein interactions. We hypothesize that the CoREST complex will process multiply-modified histone tails more efficiently that the LSD1 and HDAC1 enzymes acting on their own. Aim 1 will focus on substrate specificity by evaluating the enzymatic activities of the complex on post-translationally modified peptide substrates. Aim 2 will explore active site coordination by analyzing hybrid substrate-inhibitor peptides that can dock the inhibitor moiety in the active site of HDAC1 or LSD1 and the substrate moiety in the partner enzyme. Furthermore, by varying the distance between these two groups on the same peptide, we hope to determine complex orientation, geometry and the role of protein-protein interactions in enzymatic specificity. In Aim 3, we plan to extend these studies to the more physiologically relevant nucleosome substrates engineered to have specific PTMs. These experiments can reveal whether there are special features of the CoREST complex interactions with this more natural substrate form. Gene expression regulation rarely involves single enzymes catalyzing modifications and investigating intact complexes as a whole is likely to give more accurate insight into their functions. In addition, silencing of gene expression through epigenetic mechanisms is a hallmark of cancer and other diseases, and much work from this laboratory and others has been focused on discovering novel inhibitors for LSD1 and HDACs. Therefore, studies on the biological mechanisms of these complexes can not only enhance our fundamental understanding of gene regulation but it can pave the way toward improved therapeutics.